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Resolution of racemate; |
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EXAMPLE l[0046] This example demonstrates a Reverse Phase procedure for isolating cisatracurium besylate on C18 stationary phase using different aqueous phases. [0047] Atracurium besylate reference sample ( 10 mg/ml) was analyzed on a C 18 stationary phase by gradient elution, using different aqueous phases in the solvent mixtures with methanol. Column and packing : Alltech, Altima C18, 250x4.6x5μ, Cat. No. 88056; UV detection: 280 nm; flow rate: 1 ml/min; The results, including the gradient elutions, are detailed in Table 4: Table 4and w1?W2 are the corresponding widths at the bases of the peaks obtained by extrapolating the relatively straight sides of the peaks to the baseline. EXAMPLE 2[0048] This example demonstrates the comparison of acetate and formate buffers for RP chromatographic separations of (lR,l'R)-atracurium besylate isomers. [0049] (IR, l'R)-atracurium besylate finished dosage form (the reference sample), having concentration of 10 mg/ml, was analyzed on a C18 stationary phase by gradient elution, using a mixture of methanol and a buffer (pH 3.5). Two buffers were compared: acetate buffer and formate buffer (both of them are prepared using the corresponding ammonia salt). The results are presented in Table 5.Table 5min=minutes EXAMPLE 3[0050] This example demonstrates the effect of pH on RP chromatographic separations of of (lR,rR)-atracurium besylate isomers.[0051] (IR, l'R)-atracurium besylate finished dosage form (the reference sample), having concentration of 10 mg/ml, was analyzed on a Cl 8 stationary phase by gradient elution using a mixture of methanol and a buffer. The pH of the ammonium acetate buffer (20 mM) was varied from 3.0 to 5.5. The results are depicted in Table 6. Table 6min = minutes EXAMPLE 4[0052] This example demonstrates the effect of the buffer salt concentration on RP chromatographic separation of (lR,rR)-atracurium besylate isomers. [0053] (IR, l'R)-Atracurium besylate finished dosage form (the reference sample), having concentration of 10 mg/ml, was analyzed on a C 18 stationary phase by gradient elution of methanol buffer. The concentration of ammonium acetate buffer (pH=3.5) was varied from 5mM to 100 mM. The results are depicted in Table 7.Table 7min=minutes EXAMPLE 5[0054] This example demonstrates the separation of the ( IR, 1 'R)-atracurium besylate reference sample.[0055] The (IR, l'R)-atracurium besylate reference sample was separated by semi- preparative Reverse Phase HPLC method as follows: Hypersil Hyperprep HS C 18, 250x21.2 mm, 15μ Column, conditions: 20 mM NaNO3, pH adjusted to 2.0 with HNO3. Eluent B: methanol. Flow rate: 13 ml/minute. The gradient elution is as detailed in Table 2. [0056] The sample solutions for the preparative HPLC separation were prepared as follows: Solution 1, 827.3 mg of the (lR,l'R)-atracurium besylate reference sample was dissolved in 20 ml Eluent A (concentration: 33.1 mg/ml). Solution 2, 623.5 mg of the (lR,rR)-atracurium besylate reference sample was dissolved in 20 ml Eluent A (concentration. 31.2 mg/ml). A (lR,l'R)-atracurium besylate reference sample, having concentration of 1.56 mg/ml was prepared and kept cold for use in the identification and quantization of the isomers.[0057] The sample solutions for preparative separation were loaded into the Reverse Phase C18 column. The column was eluted with 20 mM NaNO3 solution (pH adjusted to 2.0 with HNO3) and methanol. Table 8 summarizes the results of 11 runs of analyses of the combined fractions.Table 8[0058] Fractions of the column eluate were collected and the fractions, containing the required lR-cis,l'R-cis (cisatracurium) isomer, were combined and analyzed against the reference solution. Table 9 summarizes the results of analyses of the (lR,l'R)-atracurium besylate isomers. As indicated in Tables 9 and 10, the total loading of (IR, I1R)- atracurium besylate was 534.4 mg, while the total loading of cisatracurium besylate was 299.4 mg and the total loading of cisatracurium base was 222.9 mg (90% yield). Table 9Table 10 EXAMPLE 6[0059] This example demonstrates the stability of (IR, l'R)-atracurium besylate in different buffers and at different temperatures.[0060] The stability of the (lR,rR)-atracurium besylate solution at room temperature for time periods of up to 24 hours was checked using different types of buffers (varying by the nature of the cation and the anion). The diluent was a mixture of 90% buffer and 10% methanol. The Cation concentration in each buffer was 20 mM. The HPLC conditions were according to the USP procedure. The results are depicted in Tables 11 and 12. The degradation (D), according to the data presented in Table 11, was calculated as follows:Xo- X24D = X lOOXo wherein, X0 = % of cisatracurium at To, and X24 = % of cisatracurium at after 24 hours. Table 11A graph depicting the stability of the lR-cis, lR'-trans isomer at different pH values is provided in Fig. 7, which demonstrates that at pH 3, after 20 hours the % area of the IR- cis,l'R-cis isomer is only slightly reduced while at pH 5.5 the % area of the lR-cis, l'R-cis isomer is significantly reduecd.[0061] The degradation (D) according to the data presented in Table 12 was calculated as follows: D = X 100Xo wherein, X0 = % of cisatracurium at T0, and X2] = % of cisatracurium at after 24 hours.Table 12*The buffer used was the Na CH3COOYCH3COOH buffer at 3 different pH values, that is pH values of 3.0, 4.6 and 5.5. The values in the table are represented as % of cisatracurium besylate.A sample solution of (lR,l'R)-atracurium besylate (10 mg/ml) was prepared using two buffer solutions at pH values of 1.0 and 2.0 and analyzed on the C18 stationary phase by gradient elution [2OmM KNO3 buffer (at pH corresponding to sample preparation) - methanol]. The stability of the sample solution at the mentioned pH values was demonstrated at room temperature and at 4°C, as depicted in Table 13.Table 13RT= room temperature, D=degradationXo- X 6/26D = X 100Xo wherein, X 0 = % of cisatracurium at T0, and X 6/26 = % of cisatracurium at after 6 or 26 hours.EXAMPLE 7[0062] This example demonstrates a method for purification of the cisatracurium solution from the buffer's mixture by Solid Phase Extraction (SPE). [0063] A series of the sample solutions of (lR,l'R)-atracurium besylate isomer mixture (55% cis-cis; 35% cis-trans and 6% trans-trans isomer) was prepared in diluents containing different buffers (varying by the nature of the cation and the anion). The diluents consisted of a mixture of 90% buffer and 10% methanol. The sample solutions were purified using SPE C18 cartridge.[0064] The evaluation of the buffer anions was carried out by HPLC. The cations were evaluated indirectly. The recovery of the isolate (IR cis.l'R-cis isomer) and anions was checked after each step of the SPE method, which comprises the steps of:1) successive transferring of the sample solution and water through the sorbent;2) elution of the sample with methanol; and3) washing the sorbent with methanol.The results of this study are summarized in the Table 14. Table 14ND-Not detected, NE-Not evaluated EXAMPLE 8[0065] This example demonstrates a method of product isolation. [0066] Fractions of column eluates containing the lR-cis,l'R-cis isomer were collected manually viaHypersil Hyperprep HS C18 column, 250mm*21.2mm*15μ, P/N 37115-125, using the following eluents:Eluent A: 2OmM NaNO3 aqueous solution, pH adjusted to 2.0 with HNO3 Eluent B: methanol.The gradient was as described in Table 3, and the detection was at 280 nm. The flow rate was 14 ml/min and the cistracurium besylate was isolated from the (lR,rR)-atracurium besylate mixture and analyzed using an HPLC system. The Fractions were combined correspondingly to the lR-cis,l'R-cis isomer content, as detailed in Table 15.Table 15The fractions were combined (400 ml) and mixed with 200 ml of acidified brine (pH=2 with benzenesulfonic acid) and extracted with 150 ml dichloromethane (three consecutive extractions, 50 ml of dichloromethane each extraction). The organic phases were collected, dried with MgSO4 and evaporated to dryness to afford residual semi-solid oil (91 mg), which was dissolved in 18 ml water and the pH was adjusted to ~3 with benzenesulfonic acid. The aqueous solution was placed into the freeze dryer (in tree glass vials) for 40 hours. The aqueous solution was lyophilized to afford 72 mg of cisatracurium besylate in 60% yield, having purity of 96.3% (by HPLC). |